Electrode Surface Potential-Driven Protein Adsorption and Desorption through Modulation of Electrostatic, van der Waals, and Hydration Interactions.

When proteins in aqueous solutions are exposed to solid substrates, they adsorb due to the dynamic interplay of electrostatic, van der Waals, and hydration interactions and do so in a rather irreversible fashion, which makes protein recovery troublesome. Here, we use a gold electrode as the solid substrate and modulate the surface potential to systematically induce protein adsorption as well as partial desorption. We use different methods such as surface plasmon resonance, atomic force microscopy, and electrowetting and show that biasing the electrode to more negative potentials (by -0.4 V compared to the open-circuit potential at pH 6) results in an increased adsorption barrier of 6 kJ mol-1 for the negatively charged protein ß-lactoglobulin. Further, we clearly demonstrate that this is due to an increased double layer potential of -0.06 V and an increase in hydration repulsion. This indicates that an electric potential can directly influence surface interactions and thus induce partial ß-lactoglobulin desorption. These observations can be the basis for biosensors as well as separation technologies that use only one trigger to steer protein ad- and desorption, which is low in energy requirement and does not generate large waste streams, as is the case for standard protein separation technologies.

Effect of pre-treatment on in vitro gastric digestion of quinoa protein (Chenopodium quinoa Willd.) obtained by wet and dry fractionation.

Quinoa protein was isolated from quinoa seeds using wet fractionation that resulted in a protein isolate (QPI) with a high protein purity of 87.1% (w/dw) and a protein yield of around 54%, and a dry fractionation method delivered a quinoa protein concentrate (QPC) with a purity of 27.8% (w/dw) and yield of around 47%. The dry fractionation process only involves milling and sieving and keeps the protein in its natural, native state. The aim was to study the in vitro gastric digestibility of both protein. Attention was paid to thermal pre-treatment of QPI and QPC. QPC showed significantly higher (p < .05) digestibility than QPI samples. The results were interpreted with a simple double exponential model. The fraction of easily digested protein in QPC is higher than for QPI. The better digestibility of the QPC was explained by the prevention of the formation of large aggregates during pre-heating of the protein.

Interfacial properties and emulsification performance of thylakoid membrane fragments.

Thylakoids membranes are sophisticated, dynamic structures found in plant leaves, composed of protein complexes in a dynamic lipid matrix. The interfacial absorption dynamics and viscoelasticity of thylakoid membranes fragments were measured to assess the properties of the interfacial layer and to elucidate an emulsifying mechanism that includes the role of thylakoid's composition and 3D structure. Thylakoid membranes were extracted from sugar beet leaves by a series of buffer washing, filtration and centrifugation. The extract containing the intact thylakoid membranes was suspended in water through high-pressure homogenisation, which disrupted the structure into membrane fragments. Thylakoid fragments showed surface and interfacial behaviour similar to soft particles or Pickering stabilizers with slow adsorption kinetics. After adsorption, an elastic and stable thin film was formed, indicating formation of new interactions between adjacent thylakoid fragments. In an emulsion, thylakoid fragments stabilised oil droplets against coalescence, despite droplet aggregation occurring already during emulsification. Droplet aggregation occurred by steric and electrostatic bridging, which in turn forms a 3D network where the oil droplets are immobilised, preventing further droplet coalescence or aggregation. It was concluded that both composition and structure of thylakoid fragments determine their emulsifying properties, conferring potential for encapsulation systems, where the search for natural materials is gaining more attention.

Cross-flow deep fat frying and its effect on fry quality distribution and mobility.

Conventional industrial frying systems are not optimised towards homogeneous product quality, which is partly related to poor oil distribution across the packed bed of fries. In this study we investigate an alternative frying system with an oil cross-flow from bottom to top through a packed bed of fries. Fluidization of rectangular fries during frying was characterised with a modified Ergun equation. Mixing was visualized by using two coloured layers of fries and quantified in terms of mixing entropy. Smaller fries mixed quickly during frying, while longer fries exhibited much less mixing, which was attributed to the higher minimum fluidization velocity and slower dehydration for longer fries. The cross-flow velocity was found an important parameter for the homogeneity of the moisture content of fries. Increased oil velocities positively affected moisture distribution due to a higher oil refresh rate. However, inducing fluidization caused the moisture distribution to become unpredictable due to bed instabilities.

Factors impeding enzymatic wheat gluten hydrolysis at high solid concentrations.

Enzymatic wheat gluten hydrolysis at high solid concentrations is advantageous from an environmental and economic point of view. However, increased wheat gluten concentrations result in a concentration effect with a decreased hydrolysis rate at constant enzyme-to-substrate ratios and a decreased maximum attainable degree of hydrolysis (DH%). We here identified the underlying factors causing the concentration effect. Wheat gluten was hydrolyzed at solid concentrations from 4.4% to 70%. The decreased hydrolysis rate was present at all solid concentrations and at any time of the reaction. Mass transfer limitations, enzyme inhibition and water activity were shown to not cause this hydrolysis rate limitation up to 50% solids. However, the hydrolysis rate limitation can be, at least partly, explained by a second-order enzyme inactivation process. Furthermore, mass transfer impeded the hydrolysis above 60% solids. Addition of enzyme after 24 h at high solid concentrations scarcely increased the DH%, suggesting that the maximum attainable DH% decreases at high solid concentrations. Reduced enzyme activities caused by low water activities can explain this DH% limitation. Finally, a possible influence of the plastein reaction on the DH% limitation is discussed.

Food proteins from yeast-based precision fermentation: Simple purification of recombinant ß-lactoglobulin using polyphosphate.

Proteins produced through precision fermentation are often purified through chromatographic methods. Faster and more cost-effective purification methods are desired for food application. Here, we present a simple method for purification of protein produced from yeast, using ß-lactoglobulin secreted from Pichia pastoris as an example. The food-grade salt hexametaphosphate (HMP) was used to precipitate the protein at acidic pH, while the impurities (extracellular polysaccharides; mainly mannan) remained soluble. After re-solubilization of the protein-HMP complex by neutralization, excess HMP was selectively precipitated using calcium chloride. The protein content of the crude sample increased from 26 to 72 wt% (comparable to purification with anion exchange chromatography), containing only residual extracellular polysaccharides (9 wt%) and HMP (1 wt%). The established method had no significant impact on the structural and functional properties (i.e., ability to form emulsions) of the protein. The presented method shows potential for cost-effective purification of recombinant proteins produced through yeast-based expression systems.

Single droplet drying with stepwise changing temperature-time trajectories: Influence on heat sensitive constituents.

Optimization procedures for industrial spray drying processes mainly rely on empirical understanding. Mechanistic understanding of the process is limited, but can be enhanced by studying the drying of single droplets. We here report on a new sessile single droplet drying platform, using two air streams to represent the inlet and outlet air of a spray dryer to simulate changing conditions in a spray dryer. Accurate temperature measurements confirmed the temperature profiles and their imposition onto a drying droplet. Single droplets of solutions containing ß-galactosidase and maltodextrin were dried with different temperature-time trajectories, with the inactivation of the enzyme as indicator for the thermal load on the droplet. The locking point is found to be an important parameter: the air temperature before this point does not influence the enzyme inactivation much, but a high air temperature after the locking point results in significant inactivation. The ß-galactosidase inactivation was also successfully predicted with a coupled drying and inactivation model.

Mild Fractionation for More Sustainable Food Ingredients.

With the rising problems of food shortages, energy costs, and raw materials, the food industry must reduce its environmental impact. We present an overview of more resource-efficient processes to produce food ingredients, describing their environmental impact and the functional properties obtained. Extensive wet processing yields high purities but also has the highest environmental impact, mainly due to heating for protein precipitation and dehydration. Milder wet alternatives exclude, for example, low pH-driven separation and are based on salt precipitation or water only. Drying steps are omitted during dry fractionation using air classification or electrostatic separation. Benefits of milder methods are enhanced functional properties. Therefore, fractionation and formulation should be focused on the desired functionality instead of purity. Environmental impact is also strongly reduced by milder refining. Antinutritional factors and off-flavors remain challenges in more mildly produced ingredients. The benefits of less refining motivate the increasing trend toward mildly refined ingredients.

Dextrose equivalence of maltodextrins determines particle morphology development during single sessile droplet drying.

Particle morphology development during spray drying is critical to powder properties. The aim of this study was to investigate whether the dextrose equivalence (DE) of maltodextrins can be used as an indicator for the final particle morphology. Maltodextrins were characterized on glass transition temperature (Tg) and viscosity, where low DE-value maltodextrins exhibited higher Tg and viscosity than high DE maltodextrins (≥21). A new custom-built sessile single droplet dryer was used to analyse morphology development of minute maltodextrin droplets (R0 ~ 100 µm) at 60 °C and 90 °C. Droplets with low DE showed early skin formation (2-5 s) and developed smoothly shaped particles with large cavities. Rheology on low DE maltodextrin films at dry matter of 82% (w/w) suggested that drying droplets acquired elasticity after locking providing resistance against surface compression. After locking morphology development is probably halted as the glassy state is approached. On the contrary, rheology on high DE maltodextrin (≥21) films at dry matter of 93% (w/w) suggested that drying droplets with high DE developed viscous skins, which are susceptible to surface deformations, leading to wrinkling, folding or creasing particle morphologies. The results demonstrated that DE-value may be used as an indicator for particle morphology development when interpreted in view of the process conditions.

Visualizing the hydrodynamics in sieve-based lateral displacement systems.

Deterministic lateral displacement (DLD) systems structure suspension flow in so called flow lanes. The width of these flow lanes is crucial for separation of particles and determines whether particles with certain size are displaced or not. In previous research, separation was observed in simplified DLD systems that did not meet the established DLD geometric design criteria, by adjusting the outflow conditions. We here investigated why these simplified DLD systems are able to displace particles, by experimentally investigating the hydrodynamics in the device. Flow lanes were visualized and the local flow velocities were measured using µPIV and compared with 2D fluid dynamics simulations. The size of the flow lanes strongly correlates with the local flow velocity (Vy and Vx), which depends on the hydrodynamics. Therefore, the geometric design criteria of DLD devices is in fact just one method to control the local hydrodynamics, which may also be influenced by other means. These findings give a new perspective on the separation principle, which makes the technique more flexible and easier to translate to industrial scale.

Morphology development during single droplet drying of mixed component formulations and milk.

We report on the influence of selected components and their mixtures on the development of the morphology during drying of single droplets and extend the results to the morphology of whole milk powder particles. Sessile single droplet drying and acoustic levitation methods were employed to study single droplet drying. The influence of carbohydrates (lactose and maltodextrin DE12) and proteins (micellar casein or whey protein) on morphology development is very different, since upon concentration protein systems will jam and undergo a colloidal glass transition, whereas carbohydrate systems will gradually increase in viscosity as a consequence of the concentration. Whey protein gives relatively rigid shells due to jamming of the "hard sphere" proteins, while casein micelles behave as "soft spheres" that can deform after jamming, which gives flexibility to the shell during drying. The influence of the carbohydrates on the final morphology was found much larger than the influence of the proteins. Caseins influenced morphology only in mixtures with lactose at higher concentrations due to its high voluminosity. Similar observations were done for whole milk, where fat appeared to have no influence. With maltodextrin the influence of the casein was again observed in the shape and smoothness of wrinkles. Both sessile and levitated droplet drying methods provide a similar and consistent view on morphology development.

Micrometer-sized fibrillar protein aggregates from soy glycinin and soy protein isolate.

Long, fibrillar semiflexible aggregates were formed from soy glycinin and soy protein isolate (SPI) when heated at 85 degrees C and pH 2. Transmission electron microscopy analysis showed that the contour length of the fibrils was approximately 1 microm, the persistence length 2.3 microm, and the thickness a few nanometers. Fibrils formed from SPI were more branched than the fibrils of soy glycinin. Binding of the fluorescent dye Thioflavin T to the fibrils showed that beta-sheets were present in the fibrils. The presence of the fibrils resulted in an increase in viscosity and shear thinning behavior. Flow-induced birefringence measurements showed that the behavior of the fibrils under flow can be described by scaling relations derived for rodlike macromolecules. The fibril formation could be influenced by the protein concentration and heating time. Most properties of soy glycinin fibrils are comparable to beta-lactoglobulin fibrils.

Reducing the critical particle diameter in (highly) asymmetric sieve-based lateral displacement devices.

Deterministic lateral displacement technology was originally developed in the realm of microfluidics, but has potential for larger scale separation as well. In our previous studies, we proposed a sieve-based lateral displacement device inspired on the principle of deterministic lateral displacement. The advantages of this new device is that it gives a lower pressure drop, lower risk of particle accumulation, higher throughput and is simpler to manufacture. However, until now this device has only been investigated for its separation of large particles of around 785 µm diameter. To separate smaller particles, we investigate several design parameters for their influence on the critical particle diameter. In a dimensionless evaluation, device designs with different geometry and dimensions were compared. It was found that sieve-based lateral displacement devices are able to displace particles due to the crucial role of the flow profile, despite of their unusual and asymmetric design. These results demonstrate the possibility to actively steer the velocity profile in order to reduce the critical diameter in deterministic lateral displacement devices, which makes this separation principle more accessible for large-scale, high throughput applications.

Enzyme distribution and matrix characteristics in biocatalytic particles.

In a study of Assemblase, an industrial immobilized penicillin-G acylase, various electron microscopic techniques were used to relate intra-particle enzyme heterogeneity with the morphological heterogeneity of the support material at various levels of detail. Transmission electron microscopy was used for the study of intra-particle penicillin-G acylase distribution in Assemblase particles of various sizes; it revealed an abrupt increase in enzyme loading at the particle surface (1.4-fold) and in the areas (designated halo's) surrounding internal macro-voids (7.7-fold). Cryogenic field-emission scanning electron microscopy related these abrupt local enzyme heterogeneities to local heterogeneity of the support material by revealing the presence of dense top layers surrounding both the particle exterior and the internal macro-voids. Furthermore, it showed a very distinct morphological appearance of the halo. Most probably, all these regions contained relatively more chitosan than gelatin (the polymers Assemblase was constructed of), which suggested local polymer demixing during particle production. A basic thermodynamic line of reasoning suggested that a difference in hydrophilicity between the two polymers induced local demixing. In the future, thermodynamic knowledge on such polymer interactions resulting in matrix heterogeneity may be used as a tool for biocatalyst design.

Production of glucose syrups in highly concentrated systems.

We have investigated the hydrolysis of maltodextrins in a high concentration (up to 70%), by means of enzymatic and acid catalysis. The study revealed that the equilibrium compositions of the catalyzed reactions were kinetically determined by the selectivity of the catalyst, the substrate concentration and the reaction time. A model comprising a set of two kinetic equations was used to describe the hydrolysis and condensation reactions of glucoamylase-catalyzed reactions, even to highly concentrated systems. Increased substrate concentration resulted in the formation of more condensation products. The enzyme inhibition was low and was found to be independent of the substrate concentration.

Crust morphology and crispness development during deep-fat frying of potato.

Crust formation is an important factor in determining the crispness of French fries. This study aimed at unravelling detailed structural and textural properties of the crust in relation to crispness during frying as a function of the process temperature and time. X-ray tomography showed a larger overall pore volume at higher frying times, while a lower final moisture content mainly resulted in an increase in the amount of large pores. Texture analysis revealed that the increase in porosity, due to the increased formation of pores, results in a more crispy behaviour after frying with oil of up to 180°C. At temperatures above 180°C crispness is actually found to decrease again, which is explained by the increased plastic behaviour of the crust. This may be related to the reduced glass transition temperature of the crust because of increased sugar degradation at a very high temperature.

Downstream processing of enzymatically produced geranyl glucoside.

Geraniol plays an important role in the fragrance and flavor industry. The corresponding glucoside has interesting properties as a "slow release" aroma compound. Therefore, the enzymatic production and downstream processing of geranyl glucoside were investigated. Geranyl glucoside was produced in a spray column reactor with an initial production rate of 0.58 mg x U(-1) x h(-1). A pretreated hydrophobic microfiltration membrane was used to prevent migration of the aqueous, enzyme-containing phase to the downstream process. No retention of the glucoside, which accumulated in the geraniol phase, was found. On the basis of examples from the literature, four downstream processes were tested on their viability for this system. Extraction with water and foaming were not suitable to recover geranyl glucoside from geraniol. In the first case, the glucoside selectivity for the geraniol phase was found to be high, which made extraction with water unsuccessful. In the second case it was possible to obtain a stable foam, but significant enrichment of the foam with glucoside did not occur. Adsorption on alumina and distillation under reduced pressure were applied successfully and tested in-line with the bioreactor. A maximum glucoside adsorption of 7.86 mg x g(-1) was achieved on alumina. After desorption and evaporation of the extractant the pure glucoside was obtained quantitatively. A pure product could not be obtained after distillation because a small amount of glucose was present in the permeate as well, which accumulated in the bottom fraction. It was shown that with this reactor system a production of 1 kg of geranyl glucoside in 2 days is possible using an initial amount of 50,000 units of enzyme.

Upgrading of maatjes herring byproducts: production of crude fish oil.

Fish oil has been extracted from byproducts of the maatjes (salted) herring production using a pilot plant consisting of a mincer, heat exchanger, and three-phase decanter. The crude herring oil obtained had an initial peroxide value (PV), anisidine value (AV) and free fatty acids (FFA) level of only 3 mequiv of peroxide/kg of lipid, 8.9, and 2.9%, respectively. 5,8,11,14,17-Eicosapentaenoic acid (EPA) and 4,7,10,13,16,19-docosahexaenoic acid (DHA) were present in considerable amounts (99 and 91 g/kg, respectively). During storage of the oil, no photooxidation could be detected. Storage at room temperature led to significant autoxidation over time, apparent from primary and tertiary oxidation products, measured by a decrease of hydroperoxides and an increase of fluorescent compounds (FC). Storage at 50 degrees C resulted in significant increases in secondary (AV) and tertiary oxidation (FC) products. At all storage conditions, the FFA contents remained low (<3%) and the alpha-tocopherol content remained constant. These results open the possibility for fish oil production of good quality using salted herring byproducts.

Influence of dynamic interfacial tension on droplet formation during membrane emulsification.

Membrane emulsification is a promising and relatively new technique for producing emulsions. The purpose of this study was to better understand the influence of interfacial tension on droplet formation during membrane emulsification. Droplet formation experiments were carried out with a microengineered membrane; the droplet diameter and droplet formation time were studied as a function of the surfactant concentration in the continuous phase. These experiments confirm that the interfacial tension influences the process of droplet formation; higher surfactant concentrations lead to smaller droplets and shorter droplet formation times (until 10 ms). From drop volume tensiometer experiments we can predict the interfacial tension during droplet formation. However, the strong influence of the rate of flow of the to-be-dispersed phase on the droplet size cannot be explained by the predicted values. This large influence of the oil rate of flow is clarified by the hypothesis that snap-off is rather slow in the studied regime of very fast droplet formation.

Thermozymes and their applications: a review of recent literature and patents.

Enzymes from thermophilic microorganisms, thermozymes, have unique characteristics such as temperature, chemical, and pH stability. They can be used in several industrial processes, in which they replace mesophilic enzymes or chemicals. Thermozymes are often used when the enzymatic process is compatible with existing (high-temperature) process conditions. The main advantages of performing processes at higher temperatures are reduced risk of microbial contamination, lower viscosity, improved transfer rates, and improved solubility of substrates. However, cofactors, substrates, or products might be unstable or other side reactions may occur. Recent developments show that thermophiles are a good source of novel catalysts that are of great industrial interest. Thermostable polymer-degrading enzymes such as amylases, pullulanases, xylanases, proteases, and cellulases are expected to play an important role in food, chemical, pharmaceutical, paper, pulp, and waste-treatment industries. Considerable research efforts have been made to better understand the stability of thermozymes. There are no major conformational differences with mesophilic enzymes, and a small number of extra salt bridges, hydrophobic interactions, or hydrogen bounds seem to confer the extra degree of stabilization. Currently, overexpression of thermozymes in standard Escherichia coli allows the production of much larger quantities of enzymes, which are easy to purify by heat treatment. With wider availability and lower cost, thermophilic enzymes will see more application in industry.